Deoxyuridine triphosphate nucleotidohydrolase (dUTPase) is an ubiquitous enzyme which hydrolyzes deoxyuridine triphosphate (dUTP) to deoxyuridine monophosphate (dUMP) and pyrophosphate. This reaction is thought to occur primarily to limit pools of intracellular dUTP in order to prevent significant dUMP incorporation into DNA during replication and repair. A second role of dUTPase is to provide substrate (dUMP) for the de novo synthesis of thymidylate.
In both prokaryotic and eukaryotic cell systems, dUTPase has been clearly shown to be an essential enzyme, without which the cell will die. Lack of dUTPase leads to elevated cellular dUTP pools, resulting in an increased misincorporation of dUMP into DNA.
Uracil is not a native component of DNA, and efficient repair systems have evolved to remove and repair the misincorporated base. However, due to the persistent high levels of dUTP in dUTPase.sup.- mutants, the repair of the misincorporated base by uracil-DNA glycosylase becomes a self-defeating repair process, resulting in the removal and subsequent reincorporation of dUMP. Ultimately, this process leads to DNA fragmentation and cell death. In addition to prokaryotes and eukaryotes, a number of viruses are known to encode a dUTPase function.
In the mammalian system, it has been shown that antifolate analogs and other inhibitors of de novo thymidylate biosynthesis cause an increase in the ratio of dUTP to dTTP resulting in misincorporation of dUMP, which further results in DNA fragmentation and cell death. It has been demonstrated that, in certain human tumor cell lines, increased levels of dUTPase are responsible for an increase in resistance to the cancer chemotherapeutic agent fluorodeoxyuridine (FUdR), a thymidylate synthetase inhibitor. These studies provide substantial evidence that dUTPase, the chief regulator of dUTP pools, mediates a critical step in FUdR toxicity.
Two groups of researchers, McIntosh et al., PNAS, 89:8020-8024 (1992) and Strahler et al., PNAS, 90:4991-4995 (1993), have reportedly isolated the human dUTPase enzyme and characterized the enzyme by its cDNA and amino acid sequences. McIntosh reported a cDNA of 526 base pairs containing an ORF which encoded a protein of 141 amino acids and a 3' flanking sequence following the ORF. Strahler reported the identical cDNA and amino acid sequence as did McIntosh, with the exception of two additional bases at the 5' end of the cDNA and a longer 3' flanking sequence. The human dUTPase reported by both groups was found to have a high degree of homology with dUTPase from other organisms including that from yeasts, bacteria and viruses. Strahler further reported that human dUTPase exists in both phosphorylated and a non-phosphorylated forms.
dUTPase in cells and phosphorylation of dUTPase occur in a cell-cycle dependent manner. That is, the level of dUTPase in the cell and the phosphorylation of dUTPase increase over resting levels during cell proliferation.
Ki-67 (mib-1) is another human protein which has been reported to increase during cell proliferation and to provide a means for assessing the growth fraction of human cells. A monoclonal antibody directed against Ki-67 has been reported to be useful to assess the growth fraction (i.e. the number of cells in cell cycle) of normal, reactive and neoplastic tissues. Brown, D. C. and Gatter, K. C., "Monoclonal Antibody Ki-67: Its Use in Histopathology", Histopathology, 17:489-503 (1990), incorporated herein by reference. Antibody Ki-67 recognizes an antigen which is associated with the cell nucleus. Various authors have reported on the cell kinetics, some reporting the increase in antigen expression with cell progression through the cell cycle in both normal and malignant cell lines, others report the uniform expression of Ki-67 antigen throughout the cell cycle. The topographical distribution of the antigen also appears to be cell cycle dependent.
It appears that the level of expression of Ki-67 is nutritionally dependent. This apparent nutritional influence on Ki-67 antigen expression is observable in the measurement of growth fraction of tumors. This nutritional phenomenon may be responsible for the variable results obtained in the growth fraction of tumors using Ki-67.
The exact function of Ki-67 antigen is not known. However, it is known that Ki-67 antigen is not essential for cell proliferation. The proliferative activity of a tumor or tissue is determined by the growth fraction (i.e. the number of cells in cell cycle) and the time taken to complete the cell cycle. There is strong correlation between the proliferation rate of tumors and clinical outcome, hence the desirability of using Ki-67 as a proliferation marker and a prognostic indicator.
However, several practical problems which limit the effectiveness of Ki-67 in that respect have been reported. Two of these shortcomings are important. First, Ki-67 antigen expression appears to be influenced by a cell's nutritional supply. Thus, tissue taken from the central area of a large tumor, which area may be necrotic, may give an erroneous low value for the growth fraction. Secondly, most tumors consist of a heterogeneous cell population, within which there are different proliferation rates. Thus, two different samples from the same tumor may give different values.
Noteworthy is that both of these practical considerations apply equally to other means of assessing growth fraction, such as tritiated thymidine and bromodeoxyuridine.
There are other shortcomings in using Ki-67 as a prognostic indicator. The growth fraction of a cell is assessable by Ki-67, but the time it takes for the cell to complete the cell cycle is not assessable by Ki-67. As a consequence, a tumor in which nearly all cells are in cycle, but where they spend a long time completing it, would show extensive positivity for Ki-67, yet the proliferation rate would not be that large. Alternatively, a tumor in which only a minority of cells are in cycle, but where the cycle time is very short, would have a higher proliferation rate, yet have few cells showing Ki-67 positivity. Thus, a limitation of Ki-67 expression is that it provides only information about whether a cell is in cycle or not, it does not tell about cell cycle length. Further, the level of Ki-67 will fluctuate during the cell cycle, and may even disappear from cells that are cycling.
Extensive studies using Ki-67 as a prognostic indicator have been reported for lymphoproliferative diseases, central nervous system tumors, collective tissue tumors, breast disease and others. Noteworthy is that it is not possible to use antibody Ki-67 to discriminate between benign and malignant breast tumors. Research-related uses of Ki-67 antibodies have raised the possibility of using the antibody to assess potential and therapeutic benefits of an anti-proliferation drug such as medroxyprogesterone acetate, interferon-gamma on human endometrial carcinoma cell line. However, this work is still limited by the above noted shortcomings of Ki-67.
Several other antibodies have also been suggested to be useful as proliferation markers. These include anti-PCNA/cyclin, anti-PAA, C5F10, and anti-DNA polymerase .alpha.. These antibodies have not gained as widespread acceptance as has Ki-67. For example, although PCNA appears to be most abundant in proliferating cells, it is also present, often at high levels, in non-proliferating cells. Therefore PCNA often yields a false positive result for proliferation.
It is evident from this review of the prior art about Ki-67 and other proliferation markers that difficult problems exist in this field of art which have yet to be resolved and that there is a serious need for a proliferation marker that will give reliable and accurate information about the status of a cell.